Rhodopsin is the only G-protein coupled receptor for which there is a crystal structure, and it must be noted that the crystallized protein was bound to an antagonist of this receptor. Thus, although it provides important information about the length and location of the transmembrane domains, the overall structure represents that of a receptor bound to an antagonist. Using the acetylcholine muscarinic M2 receptor we shall analyze the formation of disulfide bridges between transmembrane domains and intracellular loops in an effort to identify the segments of the receptor with greatest proximity to each other. The M2 receptor was chosen because detergent extraction from membranes does not abolish ligand binding and there are several good agonists and antagonists that can bind the receptor with good affinity and selectivity. An important part of this project is the development of methodology to purify microgram quantities of tagged receptor. The long-term goal of the project is to add purified heterotrimeric G-proteins to purified receptors to investigate which amino acids of the receptor make contact with which amino acids of the G protein. We have encountered difficulties in purifying sufficient quantities of M2 receptor.[unreadable] [unreadable] The small GTPases ARF and rab are involved in the targeting on vesicles carrying membrane proteins within mammalian cells. We reported in the past that the V2 and V1a vasopressin receptors are found in different cellular organelles following ligand induced internalization. While exploring the possible role of the small GTPase ARF6 in this phenomenon it was noted that expression of the constitutively active form of ARF6 reduced the levels of surface receptors between 80 and 90%. Since the biological role of these proteins are only partially defined we explored whether their effect involved a clathrin constitutive internalization pathway or whether ARF6 played a role in delivering newly synthesized receptor from the trans Golgi network to the surface of the cell. We found the latter to be the case and documented the participation of ARF6 in the ER to Golgi transit of the V1a, V2 and M2 muscarinic receptors. Thus, we assume ARF6 plays a similar role in vesicular traffic for all plasma membrane proteins that need to traverse the ER/Golgi compartments. [unreadable] [unreadable] Site directed mutagenesis of the V2 vasopressin receptor at the boundaries of the third intracellular loops results in a receptor with wild-type levels of expression, normal internalization and an unchanged affinity for Vasopressin. Nevertheless this receptor protein does not mediate adenylyl cyclase stimulation by hormone, an indicator of its inability to activate the heterotrimeric G-protein Gs. The K268 mutant V2R revealed an important role for this amino acid on receptor folding, and a close proximity between the cytoplasmic stems of TMs V and VI. [unreadable] [unreadable] During the isolation of phosphorylated peptides derived from cells expressing the epitope tagged human V2R we detected a novel phosphorylation site located in the middle of the third intracellular loop. In vitro experiments revealed the site to be a non-canonical substrate site for protein kinase A activity that was predicted by the available motif-analysis software only under low stringency analysis. It remains to be determined whether phosphorylation of this site modulates V2R function in the kidney principal cell where it mediates the antidiuretic effect of AVP.